Hemicellulose, second only to cellulose in abundance on earth, consists of xylan as the main constituent. Xylan is a hetero-polymer possessing .beta.-1,4 linked xylose units as a backbone with side chains containing pentose and hexose, and acetyl groups. Of these groups some of the arabinoses are esterified by p-coumaric and ferulic acids of lignin (Hartley, R. D., "Phenolic monomers and dimers of plant cell wall and their effects on fiber utilization," In: Microbial and plant opportunities to improve lignocellulosic utilization by ruminants, Akin, D. E. et al. (eds.) Elsevier Sci. Publ., Inc., New York (1990) pp. 183-193). Enzymes, particularly xylanase (EC3.2.1.8) which breaks the backbone of xylan, have received great attention for application in industries such as pulp bleaching, Eriksson, K-E. L., "Swedish developments in biotechnology related to the pulp and paper industry," (1985) TAPPI (Tech. Assoc. Pulp Pap. Ind.) 68:46-55; Jurasek, L. and Paice, M. G., "Biological bleaching of pulp", In: International pulp bleaching conference, TAPPI, Atlanta, Ga. (1988) pp. 11-13; Kantelinen, A. et al., "Hemicellulases and their potential role in bleaching," In: International pulp bleaching conference, TAPPI, Atlanta, Ga. (1988) pp. 1-9; No e, P. et al., "Action of xylanases on chemical pulp fibers. Part II: Enzymatic beating," J. Wood Sci. Technol. (1986) 6:167-184; and Yang, J. L. et al., "The impact of xylanase on bleaching of kraft pulps," TAPPI J. (1992) 75:95-101), pretreatment of animal feed (Wong, K. et al., "Multiplicity of .beta.-1,4-xylanase in microorganisms. Functions and applications," Microbiol. Rev. (1988) 52:305-317), food processing (Biely, P., "Microbial xylanolytic system," Trends Biotechnol. (1985) 3:286-290), and conversion of lignocellulose into feedstock chemicals and fuels (Jeffries, T. W., "Emerging technology for fermenting D-xylose," Trends Biotechnol. (1985) 3:208-212; Mohandas, D. V. et al., "Development of xylose-fermenting yeasts for ethanol production at high acetic acid concentrations," Sixteenth Symp. on Biotech. for Fuels and Chemicals, Gatlinburg, Tenn. (1994) Paper 16; Lu, Z. and Tsao, G. T., "Fermentation of xylose to glycerol by fungi," Sixteenth Symp. on Biotech. for Fuels and Chemicals, Gatlinburg, Tenn. (1994) Poster 35). One of the challenges for applying xylanase to the above processes is to produce large quantities of highly active enzymes at low cost.
Enzymatic conversion of xylan to its monomeric components requires the participation of several enzymes including xylanase (EC3.2.1.8), .beta.-xylosidase (EC3.2.1.37), .alpha.-L-arabinofuranosidase (EC3.2.1.55), .alpha.-glucuronidase (EC3.2.1.1), acetyl xylan esterase (EC3.1.1.6) as well as p-coumaroyl and feruloyl esterases (Borneman, W. S. et al., "Feruloyl and p-coumaroyl esterases from the anaerobic fungus Neocallimstix MC-2: Properties and functions in plant cell wall degradation," In: Hemicellulose and Hemicellulases, (M. P. Coughlan and G. Hazelwood, Eds., Portland Press, Cambridge, U.K.) (1993) pp. 85-102; Castanares, A. et al., "Purification and properties of a feruloyl and p-coumaroyl esterase from the fungus Penicillium pinophilum," Enzyme Microb. Technol. (1992) 14:875-884; Christov, L. P. and Prior, B. A., "Esterases of xylan-degrading microorganisms: Production, properties, and significance," Enzyme Microb. Technol. (1993) 15:460-475; Eriksson, K.-E. L. et al., "Microbial and enzymatic degradation of wood and wood components," Springer-Verlag, New York (1990)).
Xylanases are the key enzymes for the breakdown of xylan since they depolymerize the backbone. They have broad potential applications in wood biopulping (Eriksson, K.-E. L., "Swedish developments in biotechnology related to the pulp and paper industry," TAPPI (1985) 68:46-55; Eriksson, K.-E. L., and Kirk, T. K., "Biopulping, biobleaching and treatment of kraft bleaching effluents with white-rot fungi," In: Comprehensive biotechnology, C. W. Robinson (Ed.), Pergamon Press, Toronto (1985) 3:271-294; and Myers, G. C. et al., "Fungal pretreatment of aspen chips improves strength of refiner mechanical pulp," TAPPI (1988) 71:105-108), pulp bleaching (Jurasek, L. and Paice, M. G., "Biological bleaching of pulp", In: International pulp bleaching conference, TAPPI, Atlanta, Ga. (1988) pp. 11-13; Kantelinen, A. et al., "Hemicellulases and their potential role in bleaching," In: International pulp bleaching conference, TAPPI, Atlanta, Ga. (1988) pp. 1-9; No e, P. et al., "Action of xylanases on chemical pulp fibers. Part II: Enzymatic beating," J. Wood Sci. Technol. (1986) 6:167-184; Yang, J. L. et al., "The impact of xylanase on bleaching of kraft pulps," TAPPI J. (1992) 75:95-101), pretreatment of animal feed (Wong, K. K. Y. et al., "Multiplicity of .beta.-1,4-xylanase in microorganisms. Functions and applications," Microbiol. Rev. (1988) 52:305-317), food processing (Biely, P., "Microbial xylanolytic system," Trends Biotechnol. (1985) 3:286-290), and for the conversion of lignocellulosic material into industrial feedstock chemicals and fuels (Eriksson, K.-E. L., "Swedish developments in biotechnology related to the pulp and paper industry," TAPPI (1985) 68:46-55; Jeffries, T. W., "Emerging technology for fermenting D-xylose," Trends Biotechnol. (1985) 3:208-212).
Considering the industrial potentials of xylanases, an important aspect of xylanase research is to obtain highly active xylanases at low cost. Consequently several bacteria and fungi have been screened for xylanolytic activity (Eriksson, K.-E. L. et al., "Microbial and enzymatic degradation of wood and wood components," Springer-Verlag, New York (1990); Gilkes, N. R. et al., "Domains in microbial .beta.-1,4-glycanase: Sequence conservation, function, and enzyme families," Microbiol. Rev. (1991) 55:303-315). What has become evident is that these microorganisms produce multiple xylanases with varying specific activities.
The fungus Aureobasidium pullulans Y-2311-1 has been shown to produce the highest levels of xylanase among several xylanolytic fungi (Leathers, T. D., "Color variants of Aureobasidium pullulans overproduce xylanase with extremely high specific activity," Appl. Environ. Microbiol. (1986) 52:1026-1030; Leathers et al., "Induction and glucose repression of xylanase from a color variant strain of Aureobasidium pullulans," Biotechnol. Lett. (1986) 8:867-872; Leathers et al., "Overproduction and regulation of xylanase in Aureobasidium pullulans and Cryptococcus albidus", Biotechnol. Bioeng. Symp. (1984) 14:225-250). Unfractionated extracellular xylanase from this fungus has been used successfully for the bleaching of kraft pulps (Yang et al., "The impact of xylanase on bleaching of kraft pulps," TAPPI (1992) 75:95-101). D-Xylose, xylobiose, xylan, and arabinose all induced, while glucose repressed, xylanase activity (Leathers, T. D. et al., "Induction and glucose repression of xylanase from a color variant strain of Aureobasidium pullulans," Biotechnol. Lett. (1986) 8:867-872). Leathers (Leathers, T. D., "Color variants of Aureobasidium pullulans overproduce xylanase with extremely high specific activity," Appl. Environ. Microbiol., (1986) 52:1026-1030) showed that two xylanases with similar molecular masses were secreted into the culture supernatant by A. pullulans grown on xylan or xylose, and one of these, which we designated APX-I and which had high specific activity toward oat spelt xylan (OSX), was purified (Leathers, T. D., "Amino acid composition and partial sequence of xylanase from Aureobasidium", Biotechnol. Lett. (1988) 10:775-780; Leathers, T. D., "Purification and properties of xylanase from Aureobasidium," J. Ind. Microbiol. (1989) 4:341-348).
Other organisms which produce xylanases include Streptomyces lividans (Kluepfel, D., et al., "Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66," Biochem. J. (1990) 267:45-50); Thermoascus aurantiacus (Shepherd, M. G. et al., "Substrate specificity and mode of action of the cellulases from the thermophilic fungus Thermoascus aurantiacus," Biochem J. (1981) 193:67-74); Thermotoga sp. strain Fj SS3-B.1 (Simpson, H. D. et al., "An extremely thermostable xylanase from the thermophilic eubacterium Thermotoga," Biochem. J. (1991) 227:413-417); Penicillium capsulatum and Talaromyces emersonii (Filho, E. X. et al., "The xylan-degrading enzyme systems of Penicillium capsulatum and Talaromyces emersonii," Biochem. Soc. Trans. (1991) 19:25S); Caldocellum saccharolyticum (Luthi, E. et al., "Cloning, sequence analysis, and expression of genes encoding xylan-degrading enzymes from the thermophile Caldocellum saccharolyticum," Appl. Environ. Microbiol. (1990) 56:1017-1024); Bacillus stearothermophilus (Gat, O. et al, "Cloning and DNA sequence of the gene coding for Bacillus stearothermophilus T-6 xylanase," Appl. Environ. Microbiol. (1994) 60:1889-1896); and Thermonospora fusca (Ghangas, G. S. et al., "Cloning of a Thermonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans," J. Bacteriol. (1989) 171:2963-2969).
Yeast (Saccharomyces cerevisiae) has been widely used as a host organism for the production of heterologous proteins such as enzymes, structural proteins, hormones, interferons, and cytokines (Collins, S. H., "Production of Secreted Proteins in Yeast," in Protein Production by Biotechnology (Harris, T. J. R. ed.), Elsevier (1990) 61-77; Hitzeman, R. A. et al., "Expression of a human gene for interferon in yeast," Nature (London) (1981) 293:717-722; Innis, M. A. et al., "Expression, glycosylation, and secretion of an Aspergillus glucoamylase by Saccharomyces cerevisiae", Science (1985) 228:21-26; Marten and Seo (1991) "Engineering studies of protein secretion in recombinant Saccharomyces cerevisiae," In: Expression Systems and Processes for rDNA Products (Hatch, R. T., et al. eds.); Kniskern, P. J. et al., "Constitutive and regulated expression of the hepatitis B virus (HBV) preS2+S protein in recombinant yeast," In: Expression systems and processes for rDNA products, R. T. Hatch, C. Goochee, A. Moreira and Y. Alroy (eds.) 1991; and Demolder, J. et al., "Efficient synthesis of secreted murine interleukin -2 by Saccharomyces cerevisiae: influence of the 3' untranslated regions and codon usage," Gene (1992) 111:207-213). A xylanase gene from Cryptococcus albidus has been expressed in S. cerevisiae (Moreau, A. et al., "Secretion of a Cryptococcus albidus xylanase in Saccharomyces cerevisiae", Gene (1992) 116:109-113). Unlike bacteria, yeast does not produce endotoxins, and products from yeast are considered safe for uses in pharmaceutical and food products. Another advantage of using yeast as a host organism for heterologous protein production is that large-scale production and downstream processing of the organism and its products are readily established considering that this organism is the most commonly used organism for fermentation. Moreover, with the advance of molecular biology, genetic manipulation of yeast has become as routine as genetic manipulation of bacteria. Furthermore, most pharmaceutically and industrially important eukaryotic proteins require post-translational modifications during translocation through the endoplasmic reticulum (ER) and cell membrane. These modifications include proper folding, glycosylation, disulfate bond formation, and proteolysis. Yeast has a secretion system similar to higher eukaryotes. Most importantly, proteins secreted into yeast culture medium are protected from aggregation and protease degradation and more easily purified since yeast itself does not secrete a lot of proteins into culture medium.
Other organisms have been used for expression of foreign xylanase genes. A B. subtilis xylanase gene was expressed in B. cereus and used for pretreatment of pulp in a papermaking process. (Tremblay, L. and Archibald, F., "Production of a cloned xylanase in Bacillus cereus and its performance in Kraft pulp prebleaching," Can. J. Microbiol. (1993) 39:853-860).
Secretion of proteins is facilitated by hydrophobic-residue-rich short signal peptides on the N-terminal regions of protein precursors. Several secreted yeast proteins and peptides including invertase and mating factor .alpha. pheromone (.alpha. factor) have been shown to possess such signal peptides. These signal peptides are cleaved by specific peptidases during the secretion process. A number of heterologous proteins when fused to these yeast signal peptides are often retained in periplasmic space or secreted into culture medium at low yield (Das, R. C. and Shultz, J. L., "Secretion of heterologous proteins from Saccharomyces cerevisiae", Biotechnol. Progress (1987) 3:43-48; Marten and Seo (1991) "Engineering studies of protein secretion in recombinant Saccharomyces cerevisiae," In: Expression Systems and Processes for rDNA Products (Hatch, R. T., et al. eds.) Chaudhuri, B. et al., "The pro-region of the yeast prepro-.alpha.-factor is essential for membrane translocation of human insulin-like growth factor 1 in vivo," Eur. J. Biochem. (1992) 206:793-800).
Work upon which the present application is based in part has been published in Li, Xin-Liang et al. (1993), "Purificiation and Characterization of a New Xylanase (APX II) from the Fungus Aureobasidium pullulans Y-2311-1," Applied and Environmental Microbiology 59:3212-3218 and Li, Xin-Liang and Ljungdahl, Lars G (1994), "Cloning, Sequencing and Regulation of a Xylanase Gene from the Fungus Aureobasidium pullulans Y-2311-1," Applied and Environmental Microbiology 60:3160-3166, both of which are fully incorporated herein by reference.
All publications referred to herein are incorporated herein in their entirety.
There is a need in the art for a high-specific-activity xylanase in pure form which degrades hemicellulose, and for DNA encoding this xylanase to enable methods of producing the xylanase in pure form. There is a further need in the art for an efficient signal sequence for use in Saccharomyces cerevisiae fermentation to increase yield by increasing secretion of the product.